Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to polypeptides having cellobiohydrolase activity, catalytic domains, carbohydrate binding modules and polynucleotides encoding the polypeptides, catalytic domains or carbohydrate binding modules. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or carbohydrate binding modules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/CN2016/083574, filed May 27, 2016,which claims priority benefit under 35 U.S.C. 119 of internationalapplication no. PCT/CN2015/079983, filed May 27, 2015, and internationalapplication no. PCT/CN2015/086283, filed May 27, 2015, filed Aug. 6,2015. The contents of these applications are fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having cellobiohydrolaseactivity, catalytic domains, and carbohydrate binding modules, andisolated polynucleotides encoding the polypeptides, catalytic domains,and carbohydrate binding modules. The invention also relates to nucleicacid constructs, vectors, and host cells comprising the polynucleotidesas well as methods of producing and using the polypeptides, catalyticdomains, and carbohydrate binding modules.

Description of the Related Art

Cellulose is a polymer of glucose linked by beta-1,4-bonds. Manymicroorganisms produce enzymes that hydrolyze beta-linked glucans. Theseenzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol. Since glucose isreadily fermented to ethanol by a variety of yeasts while cellobiose isnot, any cellobiose remaining at the end of the hydrolysis represents aloss of yield of ethanol. More importantly, cellobiose is a potentinhibitor of endoglucanases and cellobiohydrolases. The accumulation ofcellobiose during hydrolysis is undesirable for ethanol production.

The present invention provides polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides having cellobiohydrolaseactivity selected from the group consisting of:

(a) a polypeptide having at least 82% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the full-length complement of (i);

(c) a polypeptide encoded by a polynucleotide having at least 82%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention also relates to polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 82% sequence identity to aminoacids 26 to 466 of SEQ ID NO: 2;

(b) a catalytic domain encoded by a polynucleotide that hybridizes underhigh stringency conditions with (i) nucleotides 76 to 1398 of SEQ ID NO:1, (ii) the full-length complement of (i);

(c) a catalytic domain encoded by a polynucleotide having at least 82%sequence identity to nucleotides 76 to 1398 of SEQ ID NO;

(d) a variant of amino acids 26 to 466 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention also relates to polypeptides comprising acarbohydrate binding module selected from the group consisting of:

(a) a carbohydrate binding module having at least 82% sequence identityto amino acids 489 to 525 of SEQ ID NO: 2;

(b) a carbohydrate binding module encoded by a polynucleotide thathybridizes under high stringency conditions with (i) nucleotides 1465 to1575 of SEQ ID NO: 1, (ii) the full-length complement of (i);

(c) a carbohydrate binding module encoded by a polynucleotide having atleast 82% sequence identity to nucleotides 1465 to 1575 of SEQ ID NO: 1;

(d) a variant of amino acids 489 to 525 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the carbohydrate binding module of (a), (b), (c), or(d) that has binding activity.

The present invention also relates to polynucleotides encoding thepolypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellobiohydrolase activity of the present invention. In one aspect, theprocesses further comprise recovering the degraded or convertedcellulosic material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellobiohydrolase activity of thepresent invention. In one aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another aspect, theprocesses further comprise recovering the fermentation product from thefermentation.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO: 2,which is operably linked to a gene encoding a protein; nucleic acidconstructs, expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA map of p505-GH7__Hami.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esteraseactivity can be determined using 0.5 mM p-nitrophenylacetate assubstrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esteraseis defined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase.Alpha-L-arabinofuranosidase activity can be determined using 5 mg ofmedium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5in a total volume of 200 μl for 30 minutes at 40° C. followed byarabinose analysis by AMINEX® HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. Alpha-glucuronidase activity can be determined according to deVries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidaseequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Auxiliary Activity 9 polypeptide: The term “Auxiliary Activity 9polypeptide” or “AA9 polypeptide” means a polypeptide classified as alytic polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl.Acad. Sci. USA 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol.6: 1399-1406; Lin et al., 2012, Structure 20: 1051-1061). AA9polypeptides were formerly classified into the glycoside hydrolaseFamily 61 (GH61) according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic material by anenzyme having cellulolytic activity. Cellulolytic enhancing activity canbe determined by measuring the increase in reducing sugars or theincrease of the total of cellobiose and glucose from the hydrolysis of acellulosic material by cellulolytic enzyme under the followingconditions: 1-50 mg of total protein/g of cellulose in pretreated cornstover (PCS), wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of an AA9polypeptide for 1-7 days at a suitable temperature, such as 40° C.-80°C., e.g., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75°C., or 80° C. and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST™ 1.5L (Novozymes A/S, Bagsværd, Denmark) and beta-glucosidaseas the source of the cellulolytic activity, wherein the beta-glucosidaseis present at a weight of at least 2-5% protein of the cellulase proteinloading. In one aspect, the beta-glucosidase is an Aspergillus oryzaebeta-glucosidase (e.g., recombinantly produced in Aspergillus oryzaeaccording to WO 02/095014). In another aspect, the beta-glucosidase isan Aspergillus fumigatus beta-glucosidase (e.g., recombinantly producedin Aspergillus oryzae as described in WO 02/095014).

AA9 polypeptide enhancing activity can also be determined by incubatingan AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

AA9 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic materialcatalyzed by enzyme having cellulolytic activity by reducing the amountof cellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

The AA9 polypeptide can be used in the presence of a soluble activatingdivalent metal cation according to WO 2008/151043 or WO 2012/122518,e.g., manganese or copper.

The AA9 polypeptide can also be used in the presence of a dioxycompound, a bicylic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic orhemicellulosic material such as pretreated corn stover (WO 2012/021394,WO 2012/021395, WO 2012/021396, WO 2012/021399, WO 2012/021400, WO2012/021401, WO 2012/021408, and WO 2012/021410).

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity can be determinedusing 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

Carbohydrate binding module: The term “carbohydrate binding module”means a domain within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose.

Catalase: The term “catalase” means ahydrogen-peroxide:hydrogen-peroxide oxidoreductase (E.C. 1.11.1.6 orE.C. 1.11.1.21) that catalyzes the conversion of two hydrogen peroxidesto oxygen and two waters.

Catalase activity can be determined by monitoring the degradation ofhydrogen peroxide at 240 nm based on the following reaction:2H₂O₂→2H₂O+O₂

The reaction is conducted in 50 mM phosphate pH 7 at 25° C. with 10.3 mMsubstrate (H₂O₂). Absorbance is monitored spectrophotometrically within16-24 seconds, which should correspond to an absorbance reduction from0.45 to 0.4. One catalase activity unit can be expressed as one μmole ofH₂O₂ degraded per minute at pH 7.0 and 25° C.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

In one aspect, the polypeptides of the present invention have at least20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 100% of thecellobiohydrolases activity of the mature polypeptide of SEQ ID NO: 2.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo1 filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in pretreated cornstover (PCS) (or other pretreated cellulosic material) for 3-7 days at asuitable temperature such as 40° C.-80° C., e.g., 40° C., 45° C., 50°C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C., and a suitablepH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,or 9.0, compared to a control hydrolysis without addition ofcellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In one aspect, the cellulosicmaterial is any biomass material. In another aspect, the cellulosicmaterial is lignocellulose, which comprises cellulose, hemicelluloses,and lignin.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another embodiment, the cellulosic material is arundo, bagasse,bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw, sugarcane straw, switchgrass, or wheat straw.

In another embodiment, the cellulosic material is aspen, eucalyptus,fir, pine, poplar, spruce, or willow.

In another embodiment, the cellulosic material is algal cellulose,bacterial cellulose, cotton linter, filter paper, microcrystallinecellulose (e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another embodiment, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Dissolved Oxygen Saturation Level: The saturation level of oxygen isdetermined at the standard partial pressure (0.21 atmosphere) of oxygen.The saturation level at the standard partial pressure of oxygen isdependent on the temperature and solute concentrations. In an embodimentwhere the temperature during hydrolysis is 50° C., the saturation levelwould typically be in the range of 5-5.5 mg oxygen per kg slurry,depending on the solute concentrations. Hence, a concentration ofdissolved oxygen of 0.5 to 10% of the saturation level at 50° C.corresponds to an amount of dissolved oxygen in a range from 0.025 ppm(0.5×5/100) to 0.55 ppm (10×5.5/100), such as, e.g., 0.05 to 0.165 ppm,and a concentration of dissolved oxygen of 10-70% of the saturationlevel at 50° C. corresponds to an amount of dissolved oxygen in a rangefrom 0.50 ppm (10×5/100) to 3.85 ppm (70×5.5/100), such as, e.g., 1 to 2ppm. In an embodiment, oxygen is added in an amount in the range of 0.5to 5 ppm, such as 0.5 to 4.5 ppm, 0.5 to 4 ppm, 0.5 to 3.5 ppm, 0.5 to 3ppm, 0.5 to 2.5 ppm, or 0.5 to 2 ppm.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity can also bedetermined using carboxymethyl cellulose (CMC) as substrate according tothe procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5,40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. Feruloyl esterase activity can be determinedusing 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetatepH 5.0. One unit of feruloyl esterase equals the amount of enzymecapable of releasing 1 μmole of p-nitrophenolate anion per minute at pH5, 25° C.

Fragment: The term “fragment” means a polypeptide or a catalytic orcarbohydrate binding module having one or more (e.g., several) aminoacids absent from the amino and/or carboxyl terminus of a maturepolypeptide or domain or module; wherein the fragment hascellobiohydrolase activity. In one aspect, a fragment contains at least425 amino acid residues, at least 450 amino acid residues, or at least475 amino acid residues of, for example, the mature polypeptide of SEQID NO: 2.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 40°C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.,and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, or 9.0.

Hemicellulosic material: The term “hemicellulosic material” means anymaterial comprising hemicelluloses. Hemicelluloses include xylan,glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. Thesepolysaccharides contain many different sugar monomers. Sugar monomers inhemicellulose can include xylose, mannose, galactose, rhamnose, andarabinose. Hemicelluloses contain most of the D-pentose sugars. Xyloseis in most cases the sugar monomer present in the largest amount,although in softwoods mannose can be the most abundant sugar. Xylancontains a backbone of beta-(1-4)-linked xylose residues. Xylans ofterrestrial plants are heteropolymers possessing abeta-(1-4)-D-xylopyranose backbone, which is branched by shortcarbohydrate chains. They comprise D-glucuronic acid or its 4-O-methylether, L-arabinose, and/or various oligosaccharides, composed ofD-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-typepolysaccharides can be divided into homoxylans and heteroxylans, whichinclude glucuronoxylans, (arabino)glucuronoxylans,(glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See,for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.Hemicellulosic material is also known herein as “xylan-containingmaterial”.

Sources for hemicellulosic material are essentially the same as thosefor cellulosic material described herein.

In the processes of the present invention, any material containinghemicellulose may be used. In a preferred aspect, the hemicellulosicmaterial is lignocellulose.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance). An isolated substancemay be present in a fermentation broth sample; e.g. a host cell may begenetically modified to express the polypeptide of the invention. Thefermentation broth from that host cell will comprise the isolatedpolypeptide.

Laccase: The term “laccase” means a benzenediol:oxygen oxidoreductase(E.C. 1.10.3.2) that catalyzes the following reaction: 1,2- or1,4-benzenediol+O₂=1,2- or 1,4-benzosemiquinone+2 H₂O.

Laccase activity can be determined by the oxidation of syringaldazine(4,4′-[azinobis(methanylylidene)]bis(2,6-dimethoxyphenol)) to thecorresponding quinone4,4′-[azobis(methanylylideneDbis(2,6-dimethoxycyclohexa-2,5-dien-1-one)by laccase. The reaction (shown below) is detected by an increase inabsorbance at 530 nm.

The reaction is conducted in 23 mM MES pH 5.5 at 30° C. with 19 μMsubstrate (syringaldazine) and 1 g/L polyethylene glycol (PEG) 6000. Thesample is placed in a spectrophotometer and the change in absorbance ismeasured at 530 nm every 15 seconds up to 90 seconds. One laccase unitis the amount of enzyme that catalyzes the conversion of 1 μmolesyringaldazine per minute under the specified analytical conditions.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 26 to 525 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 25 of SEQ ID NO: 2 are a signal peptide. TheMS analysis shows that the N-terminal of the mature polypeptide beginswith QQVGTQKAETHP, this proves the prediction of the mature polypeptideis right.

It is known in the art that a host cell may produce a mixture of two ormore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

It is also known in the art that different host cells processpolypeptides differently, and thus, one host cell expressing apolynucleotide may produce a different mature polypeptide (e.g., havinga different C-terminal and/or N-terminal amino acid) as compared toanother host cell expressing the same polynucleotide. In one aspect, amature polypeptides contains up to 505 amino acid residues (e.g., aminoacids 21 to 525 of SEQ ID NO: 2), up to 515 amino acid residues (e.g.,amino acids 11 to 525 of SEQ ID NO: 2), or up to 525 amino acid residues(e.g., amino acids 1 to 525 of SEQ ID NO: 2).

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 76 to 1575 of SEQ ID NO: 1 or the cDNAsequence thereof based on the SignalP 3.0 program (Bendtsen et al.,2004, supra) that predicts nucleotides 1 to 75 of SEQ ID NO: 1 encode asignal peptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Peroxidase: The term “peroxidase” means an enzyme that converts aperoxide, e.g., hydrogen peroxide, to a less oxidative species, e.g.,water. It is understood herein that a peroxidase encompasses aperoxide-decomposing enzyme. The term “peroxide-decomposing enzyme” isdefined herein as an donor:peroxide oxidoreductase (E.C. number1.11.1.x, wherein x=1-3, 5, 7-19, or 21) that catalyzes the reactionreduced substrate (2e⁻)+ROOR′→oxidized substrate+ROH+R′OH; such ashorseradish peroxidase that catalyzes the reactionphenol+H₂O₂→quinone+H₂O, and catalase that catalyzes the reactionH₂O₂+H₂O₂→O₂+2H₂O. In addition to hydrogen peroxide, other peroxides mayalso be decomposed by these enzymes.

Peroxidase activity can be determined by measuring the oxidation of2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) by aperoxidase in the presence of hydrogen peroxide as shown below. Thereaction product ABTS_(ox) forms a blue-green color which can bequantified at 418 nm.H₂O₂+2ABTS_(red)+2H⁺→2H₂O+2ABTS_(ox)

The reaction is conducted in 0.1 M phosphate pH 7 at 30° C. with 1.67 mMsubstrate (ABTS), 1.5 g/L TRITON® X-405, 0.88 mM hydrogen peroxide, andapproximately 0.040 units enzyme per ml. The sample is placed in aspectrophotometer and the change in absorbance is measured at 418 nmfrom 15 seconds up to 60 seconds. One peroxidase unit can be expressedas the amount of enzyme required to catalyze the conversion of 1 μmoleof hydrogen peroxide per minute under the specified analyticalconditions.

Pretreated cellulosic or hemicellulosic material: The term “pretreatedcellulosic or hemicellulosic material” means a cellulosic orhemicellulosic material derived from biomass by treatment with heat anddilute sulfuric acid, alkaline pretreatment, neutral pretreatment, orany pretreatment known in the art.

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The term “very low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.]

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase activity. In one aspect, a subsequencecontains at least 1275 nucleotides, at least 1350 nucleotides, or atleast 1425 nucleotides of, for example, the mature polypeptide codingsequence of SEQ ID NO: 1.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrimann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Reference to “about” a value or parameter herein includes aspects thatare directed to that value or parameter per se. For example, descriptionreferring to “about X” includes the aspect “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that the aspects of the invention describedherein include “consisting” and/or “consisting essentially of” aspects.

Unless defined otherwise or clearly indicated by context, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellobiohydrolase Activity

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least82%, e.g., at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which havecellobiohydrolase activity. In one aspect, the polypeptides differ by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 2.

In an embodiment, the polypeptide is isolated. In another embodiment,the polypeptide is purified.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids 26 to 525 of SEQ ID NO: 2.

In another embodiment, the present invention relates to a polypeptidehaving cellobiohydrolase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,New York). In an embodiment, the polypeptide has been isolated.

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having cellobiohydrolase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of a cell of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellobiohydrolase activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art. In one aspect, the nucleic acid probeis a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; themature polypeptide thereof; or a fragment thereof. In another aspect,the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the present invention relates to an polypeptidehaving cellobiohydrolase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding module.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant moleculesare tested for cellobiohydrolase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide. Byaligning the cellobiohydrolase shown as SEQ ID NO: 2 with known GH7polypeptides, we find one conservative active site which is 242E.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellobiohydrolase Activity

A polypeptide having cellobiohydrolase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a Hamigera polypeptide. In another aspect, thepolypeptide is a Hamigera avellanea polypeptide. In another aspect, thepolypeptide is a Hamigera fusca polypeptide. In another aspect, thepolypeptide is a Hamigera inflate polypeptide. In another aspect, thepolypeptide is a Hamigera ingelheimensis polypeptide. In another aspect,the polypeptide is a Hamigera insecticola polypeptide. In anotheraspect, the polypeptide is a Hamigera pallida polypeptide. In anotheraspect, the polypeptide is a Hamigera paravellanea polypeptide. Inanother aspect, the polypeptide is a Hamigera terricola polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

The present invention also relates to catalytic domains having asequence identity to amino acids 26 to 466 of SEQ ID NO: 2 of at least82%, e.g., at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, thecatalytic domains comprise amino acid sequences that differ by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 26to 466 of SEQ ID NO: 2.

The catalytic domain preferably comprises or consists of amino acids 26to 466 of SEQ ID NO: 2 or an allelic variant thereof; or is a fragmentthereof having cellobiohydrolase activity.

In an embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) with(i) the nucleotides 76 to 1398 of SEQ ID NO: 1, (ii) the full-lengthcomplement of (i) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 76 to 1398 of SEQ ID NO: 1 of at least 82%, e.g., at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 76 to 1398 of SEQ ID NO: 1.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 26 to 466 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids26 to 466 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or10.

Carbohydrate Binding Modules

In one embodiment, the present invention also relates to carbohydratebinding modules having a sequence identity to amino acids 489 to 525 ofSEQ ID NO: 2 of at least 82%, e.g., at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Inone aspect, the carbohydrate binding modules comprise amino acidsequences that differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from amino acids 489 to 525 of SEQ ID NO: 2.

The carbohydrate binding module preferably comprises or consists ofamino acids 489 to 525 of SEQ ID NO: 2 or an allelic variant thereof; oris a fragment thereof having carbohydrate binding activity.

In another embodiment, the present invention also relates tocarbohydrate binding modules encoded by polynucleotides that hybridizeunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions (as definedabove) with (i) the nucleotides 489 to 525 of SEQ ID NO: 1, (ii) thefull-length complement of (i) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates tocarbohydrate binding modules encoded by polynucleotides having asequence identity to nucleotides 1465 to 1575 of SEQ ID NO: 1 of atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The polynucleotide encoding the carbohydrate binding module preferablycomprises or consists of nucleotides 1465 to 1575 of SEQ ID NO: 1.

In another embodiment, the present invention also relates tocarbohydrate binding module variants of amino acids 489 to 525 of SEQ IDNO: 2 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 489 to 525 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4,5, 6, 8, 9, or 10.

A catalytic domain operably linked to the carbohydrate binding modulemay be from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase. The polynucleotideencoding the catalytic domain may be obtained from any prokaryotic,eukaryotic, or other source.

Polynucleotides

The present invention also relates to polynucleotides encoding apolypeptide, a catalytic domain, or carbohydrate binding module of thepresent invention, as described herein. In an embodiment, thepolynucleotide encoding the polypeptide, catalytic domain, orcarbohydrate binding module of the present invention has been isolated.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Hamigera, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not result in a change in the aminoacid sequence of the polypeptide, but which correspond to the codonusage of the host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions that may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. The one or more control sequences can be heterologous to thepolynucleotide.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis daI genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide. In one aspect, the cell is a Hamigera cell. In anotheraspect, the cell is a Hamigera avellanea cell. In another aspect, thecell is a Hamigera fusca cell. In another aspect, the cell is a Hamigerainflate cell. In another aspect, the cell is a Hamigera ingelheimensiscell. In another aspect, the cell is a Hamigera insecticola cell. Inanother aspect, the cell is a Hamigera pallida cell. In another aspect,the cell is a Hamigera paravellanea cell. In another aspect, the cell isa Hamigera terricola cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain or module in recoverable quantities. The polypeptide or domain ormodule may be recovered from the plant or plant part. Alternatively, theplant or plant part containing the polypeptide or domain or module maybe used as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainor module may be constructed in accordance with methods known in theart. In short, the plant or plant cell is constructed by incorporatingone or more expression constructs encoding the polypeptide or domain ormodule into the plant host genome or chloroplast genome and propagatingthe resulting modified plant or plant cell into a transgenic plant orplant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain or moduleoperably linked with appropriate regulatory sequences required forexpression of the polynucleotide in the plant or plant part of choice.Furthermore, the expression construct may comprise a selectable markeruseful for identifying plant cells into which the expression constructhas been integrated and DNA sequences necessary for introduction of theconstruct into the plant in question (the latter depends on the DNAintroduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainor module is desired to be expressed (Sticklen, 2008, Nature Reviews 9:433-443). For instance, the expression of the gene encoding apolypeptide or domain or module may be constitutive or inducible, or maybe developmental, stage or tissue specific, and the gene product may betargeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain or module in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the polynucleotide encoding a polypeptide ordomain or module. For instance, Xu et al., 1993, supra, disclose the useof the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain ormodule can be introduced into a particular plant variety by crossing,without the need for ever directly transforming a plant of that givenvariety. Therefore, the present invention encompasses not only a plantdirectly regenerated from cells which have been transformed inaccordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention. Crossing results in theintroduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain or module of the present invention comprising (a) cultivatinga transgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain or module under conditions conducive forproduction of the polypeptide or domain or module; and (b) recoveringthe polypeptide or domain or module.

Removal or Reduction of Cellobiohydrolase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby insertion, substitution, or deletion of one or more nucleotides inthe gene or a regulatory element required for transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change in the open reading frame. Such modification orinactivation may be accomplished by site-directed mutagenesis or PCRgenerated mutagenesis in accordance with methods known in the art.Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the polynucleotide to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellobiohydrolase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of the polypeptide ina cell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellobiohydrolase-free product are of particular interest in theproduction of polypeptides, in particular proteins such as enzymes. Thecellobiohydrolase-deficient cells may also be used to expressheterologous proteins of pharmaceutical interest such as hormones,growth factors, receptors, and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellobiohydrolase activity that is produced by amethod of the present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide),cell debris, biomass, fermentation media and/or fermentation products.In some embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” refers to a preparation produced bycellular fermentation that undergoes no or minimal recovery and/orpurification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP),a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme,a pectinase, a peroxidase, a protease, and a swollenin. The fermentationbroth formulations or cell compositions may also comprise one or more(e.g., several) enzymes selected from the group consisting of ahydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of uses of the compositions of the presentinvention. The dosage of the composition and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellobiohydrolase activity of the composition has been increased, e.g.,with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an AA9polypeptide, a cellulose inducible protein (CIP), a catalase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin. The compositions may alsocomprise one or more (e.g., several) enzymes selected from the groupconsisting of a hydrolase, an isomerase, a ligase, a lyase, anoxidoreductase, or a transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having cellobiohydrolase activity, orcompositions thereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition comprising a polypeptide having cellobiohydrolaseactivity of the present invention. In one aspect, the processes furthercomprise recovering the degraded cellulosic material. Soluble productsfrom the degradation of the cellulosic material can be separated frominsoluble cellulosic material using methods known in the art such as,for example, centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase activity of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme compositioncomprising a polypeptide having cellobiohydrolase activity of thepresent invention. In one aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another aspect, theprocesses further comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel (ethanol,n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals(e.g., acids, alcohols, ketones, gases, oils, and the like). Theproduction of a desired fermentation product from the cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment. In practicing the processes of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technology 100: 10-18; Mosier et al., 2005, BioresourceTechnology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on optional addition of achemical catalyst. Residence time for the steam pretreatment ispreferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes,or 4-10 minutes, where the optimal residence time depends on thetemperature and optional addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so that thecellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 2002/0164730). During steam pretreatment, hemicellulose acetylgroups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

Chemical Pretreatment. The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, Bioresource Technology 855: 1-33; Schell et al., 2004,Bioresource Technology 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng.Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, Bioresource Technology 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosicmaterial and held at a temperature in the range of preferably 140-200°C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt.% or 30-60 wt. %, such as around 40 wt. %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment. The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and/or hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically by one or more enzyme compositions in one ormore stages. The hydrolysis can be carried out as a batch process orseries of batch processes. The hydrolysis can be carried out as a fedbatch or continuous process, or series of fed batch or continuousprocesses, where the cellulosic material is fed gradually to, forexample, a hydrolysis solution containing an enzyme composition. In anembodiment the saccharification is a continuous saccharification inwhich a cellulosic material and a cellulolytic enzyme composition areadded at different intervals throughout the saccharification and thehydrolysate is removed at different intervals throughout thesaccharification. The removal of the hydrolysate may occur prior to,simultaneously with, or after the addition of the cellulosic materialand the cellulolytic enzyme composition.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s).

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the totalsaccharification time can last up to 200 hours, but is typicallyperformed for preferably about 4 to about 120 hours, e.g., about 12 toabout 96 hours or about 24 to about 72 hours. The temperature is in therange of preferably about 25° C. to about 80° C., e.g., about 30° C. toabout 70° C., about 40° C. to about 60° C., or about 50° C. to about 55°C. The pH is in the range of preferably about 3 to about 9, e.g., about3.5 to about 8, about 4 to about 7, about 4.2 to about 6, or about 4.3to about 5.5.

The dry solids content is in the range of preferably about 5 to about 50wt. %, e.g., about 10 to about 40 wt. % or about 20 to about 30 wt. %.

In one aspect, the saccharification is performed in the presence ofdissolved oxygen at a concentration of at least 0.5% of the saturationlevel.

In an embodiment of the invention the dissolved oxygen concentrationduring saccharification is in the range of at least 0.5% up to 30% ofthe saturation level, such as at least 1% up to 25%, at least 1% up to20%, at least 1% up to 15%, at least 1% up to 10%, at least 1% up to 5%,and at least 1% up to 3%. In a preferred embodiment, the dissolvedoxygen concentration is maintained at a concentration of at least 0.5%up to 30% of the saturation level, such as at least 1% up to 25%, atleast 1% up to 20%, at least 1% up to 15%, at least 1% up to 10%, atleast 1% up to 5%, and at least 1% up to 3% during at least 25%, such asat least 50% or at least 75% of the saccharification period. When theenzyme composition comprises an oxidoreductase the dissolved oxygenconcentration may be higher up to 70% of the saturation level.

Oxygen is added to the vessel in order to achieve the desiredconcentration of dissolved oxygen during saccharification. Maintainingthe dissolved oxygen level within a desired range can be accomplished byaeration of the vessel, tank or the like by adding compressed airthrough a diffuser or sparger, or by other known methods of aeration.The aeration rate can be controlled on the basis of feedback from adissolved oxygen sensor placed in the vessel/tank, or the system can runat a constant rate without feedback control. In the case of a hydrolysistrain consisting of a plurality of vessels/tanks connected in series,aeration can be implemented in one or more or all of the vessels/tanks.Oxygen aeration systems are well known in the art. According to theinvention any suitable aeration system may be used. Commercial aerationsystems are designed by, e.g., Chemineer, Derby, England, and build by,e.g., Paul Mueller Company, MO, USA.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, an AA9 polypeptide, a hemicellulase, an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin. In another aspect, the cellulase ispreferably one or more (e.g., several) enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the hemicellulase is preferably oneor more (e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. In another aspect, theoxidoreductase is preferably one or more (e.g., several) enzymesselected from the group consisting of a catalase, a laccase, and aperoxidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anAA9 polypeptide. In another aspect, the enzyme composition comprises anendoglucanase and an AA9 polypeptide. In another aspect, the enzymecomposition comprises a cellobiohydrolase and an AA9 polypeptide. Inanother aspect, the enzyme composition comprises a beta-glucosidase andan AA9 polypeptide. In another aspect, the enzyme composition comprisesan endoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase I, an endoglucanase II, or acombination of an endoglucanase I and an endoglucanase II, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises an endoglucanase I, anendoglucanase II, or a combination of an endoglucanase I and anendoglucanase II, and a beta-glucosidase. In another aspect, the enzymecomposition comprises a beta-glucosidase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase anda cellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, an AA9 polypeptide, and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase I, an endoglucanase II, or a combination of anendoglucanase I and an endoglucanase II, an AA9 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, andan AA9 polypeptide. In another aspect, the enzyme composition comprisesa beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase, anAA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase I, an endoglucanase II, or acombination of an endoglucanase I and an endoglucanase II, abeta-glucosidase, and a cellobiohydrolase I, a cellobiohydrolase II, ora combination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and an AA9 polypeptide. Inanother aspect, the enzyme composition comprises an endoglucanase I, anendoglucanase II, or a combination of an endoglucanase I and anendoglucanase II, a beta-glucosidase, an AA9 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In anembodiment, the xylanase is a Family 10 xylanase. In another embodiment,the xylanase is a Family 11 xylanase. In another aspect, the enzymecomposition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a ligninolytic enzyme. In anembodiment, the ligninolytic enzyme is a manganese peroxidase. Inanother embodiment, the ligninolytic enzyme is a lignin peroxidase. Inanother embodiment, the ligninolytic enzyme is a H₂O₂-producing enzyme.In another aspect, the enzyme composition comprises a pectinase. Inanother aspect, the enzyme composition comprises an oxidoreductase. Inan embodiment, the oxidoreductase is a catalase. In another embodiment,the oxidoreductase is a laccase. In another embodiment, theoxidoreductase is a peroxidase. In another aspect, the enzymecomposition comprises a protease. In another aspect, the enzymecomposition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and/or nativeto the host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and polypeptides havingcellobiohydrolase activity depend on several factors including, but notlimited to, the mixture of cellulolytic enzymes and/or hemicellulolyticenzymes, the cellulosic material, the concentration of cellulosicmaterial, the pretreatment(s) of the cellulosic material, temperature,time, pH, and inclusion of a fermenting organism (e.g., for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to the cellulosic material is about 0.01 toabout 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 toabout 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to cellulolytic or hemicellulolytic enzyme isabout 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g,about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g ofcellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., AA9 polypeptides can bederived or obtained from any suitable origin, including, archaeal,bacterial, fungal, yeast, plant, or animal origin. The term “obtained”also means herein that the enzyme may have been produced recombinantlyin a host organism employing methods described herein, wherein therecombinantly produced enzyme is either native or foreign to the hostorganism or has a modified amino acid sequence, e.g., having one or more(e.g., several) amino acids that are deleted, inserted and/orsubstituted, i.e., a recombinantly produced enzyme that is a mutantand/or a fragment of a native amino acid sequence or an enzyme producedby nucleic acid shuffling processes known in the art. Encompassed withinthe meaning of a native enzyme are natural variants and within themeaning of a foreign enzyme are variants obtained by, e.g.,site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, eachpolypeptide may be a Gram-positive bacterial polypeptide having enzymeactivity, or a Gram-negative bacterial polypeptide having enzymeactivity.

Each polypeptide may also be a fungal polypeptide, e.g., a yeastpolypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides mayalso be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase (GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (Gen Bank:AF487830), Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank:M15665), and Penicillium pinophilumendoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Aspergillus fumigatus cellobiohydrolase I (WO2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (Gen Bank:AY690482),Talaromyces emersonii cellobiohydrolase I (Gen Bank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

In the processes of the present invention, any AA9 polypeptide can beused as a component of the enzyme composition.

Examples of AA9 polypeptides useful in the processes of the presentinvention include, but are not limited to, AA9 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus(WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascussp. (WO 2011/039319), Penicillium sp. (emersoni0 (WO 2011/041397 and WO2012/000892), Thermoascus crustaceous (WO 2011/041504), Aspergillusaculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/113340, WO2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporusalborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477),Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO2012/135659), Humicola insolens (WO 2012/146171), Malbranchea cinnamomea(WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), andChaetomium thermophilum (WO 2012/101206), and Talaromyces thermophilus(WO 2012/129697 and WO 2012/130950).

In one aspect, the AA9 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043 or WO2012/122518, e.g., manganese or copper.

In another aspect, the AA9 polypeptide is used in the presence of adioxy compound, a bicylic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic materialsuch as pretreated corn stover (WO 2012/021394, WO 2012/021395, WO2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO2012/021408, and WO 2012/021410).

In one aspect, such a compound is added at a molar ratio of the compoundto glucosyl units of cellulose of about 10⁻⁶ to about 10, e.g., about10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 0⁻³ to about10⁻². In another aspect, an effective amount of such a compound is about0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μMto about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M,about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM toabout 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, orabout 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of an AA9 polypeptide can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and an AA9 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485),Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (Swiss Prot: Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), andTalaromyces thermophilus (GeneSeqP:BAA22816).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum(UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicolainsolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036),Myceliophtera thermophila (WO 2010/014880), Neurospora crassa(UniProt:q7s259), Phaeosphaeria nodorum (UniProt:Q0UHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt: alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (UniProt:Q96WX9), Aspergillusterreus (SwissProt:Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

Examples of oxidoreductases useful in the processes of the presentinvention include, but are not limited to, Aspergillus lentiluscatalase, Aspergillus fumigatus catalase, Aspergillus niger catalase,Aspergillus oryzae catalase, Humicola insolens catalase, Neurosporacrassa catalase, Penicillium emersonii catalase, Scytalidiumthermophilum catalase, Talaromyces stipitatus catalase, Thermoascusaurantiacus catalase, Coprinus cinereus laccase, Myceliophthorathermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinuslaccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase,Coprinus cinereus peroxidase, Soy peroxidase, Royal palm peroxidase.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (e.g., several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIO-FERM® AFT and XR (Lallemand Specialities, Inc., USA), ETHANOLREDO yeast (Lesaffre et Co, pagnie, France), FALI® (AB Mauri Food Inc.,USA), FERMIOL® (Rymco International AG, Denmark), GERT STRAND™ (GertStrand AB, Sweden), and SUPERSTART™ and THERMOSACC® fresh yeast(Lallemand Specialities, Inc., USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

In one aspect, the fermenting organism comprises a polynucleotideencoding a polypeptide having cellobiohydrolase activity of the presentinvention.

In another aspect, the fermenting organism comprises one or morepolynucleotides encoding one or more cellulolytic enzymes,hemicellulolytic enzymes, and accessory enzymes described herein.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g., pentene, hexene, heptene, andoctene); an amino acid (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone(e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionicacid, succinic acid, and xylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term“ketone” encompasses a substance that contains one or more ketonemoieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

The fungal strain NN046817 was isolated from a soil sample collectedfrom Hunan province, China, in 2000 by dilution on a YG agar plate at37° C. It was then purified by transferring a single conidium onto a YGagar plate. The NN046817 strain was identified as Hamigera sp., based onboth morphological characteristics and ITS rDNA sequence.

Media and Solutions

Minimal medium plates: 20 g agar, 20 ml Salt solution, 30 g sucrose, 100μl Triton X-100, Adjust pH to 6, add H2O to 1 liter, autoclave. Beforeuse, boil and cool down to approx. 50° C. and then add 10 ml 1Macetamide.

PDA medium contained 39 grams of potato dextrose agar and deionizedwater to 1 liter.

YG agar plates contained 5.0 g of yeast extract, 10.0 g of glucose, 20.0g of agar, and deionized water to 1 liter.

YPG medium contained 0.4% of yeast extract, 0.1% of KH2PO4, 0.05% ofMgSO4.7H2O, 1.5% glucose in deionized water.

YPM medium contained 1% yeast extract, 2% peptone and 2% maltose.

Example 1 Hamigera sp. NN046817 Genomic DNA Extraction

Hamigera sp. strain NN046817 was inoculated onto a PDA plate andincubated for 3 days at 37° C. in the darkness. Several mycelia-PDAplugs were then inoculated into 500 ml shake flasks containing 100 ml ofYPG medium. The flasks were incubated for 3 days at 37° C. with shakingat 160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem) and frozen under liquid nitrogen. Frozen mycelia wereground, by a mortar and a pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Maxi Kit (QIAGEN) following themanufacturer's instruction.

Example 2 Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Fasteris(Switzerland) for genome sequencing using an ILLUMINA® HiSeq 2000 System(Illumina, Inc.). The raw reads were assembled at Fasteris using theSOAPdenovo program (Li et al., 2010, Genome Research 20: 265-72). Theassembled sequences were analyzed using standard bioinformatics methodsfor gene identification and function prediction. GeneMark(Ter-Hovhannisyan V et al., 2008, Genome Research 18(12): 1979-1990) wasused for gene prediction. Blastall version 2.2.10 (Altschul et al.,1990, J. Mol. Biol. 215(3): 403-410 and HMMER version 2.1.1 (NationalCenter for Biotechnology Information (NCBI), Bethesda, Md., USA) wereused to predict function based on structural homology. The GH7 familycellobiohydrolase polypeptides were identified directly by analysis ofthe Blast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify start codons. SignalPprogram was further used to predict signal peptides. Pepstats (Rice etal., 2000, Trends Genet. 16(6): 276-277) was used to predict isoelectricpoints and molecular weights.

Example 3 Cloning of the Hamigera sp. GH7 Cellobiohydrolase I Gene fromGenomic DNA

The GH7 cellobiohydrolase I gene, GH7_Hami (SEQ ID NO: 1 for the genomicDNA sequence and SEQ ID NO: 2 for the deduced amino acid sequence), wasselected for expression cloning.

Based on the DNA information obtained from genome sequencing,oligonucleotide primers, shown below, were designed to amplify thecoding sequence of the GH7 cellobiohydrolase I from the genomic DNA ofHamigera sp. strain NN046817. The primers were synthesized byInvitrogen, Beijing, China.

Forward primer: (SEQ ID NO: 3) 5′-ACACAACTGGGGATCCACCatggctgctacaaaatcttaccga atctac-3′ Reverse primer: (SEQ ID NO: 4)5′-CCCTCTAGATCTCGAGacgtaacttctccagccgtctctc-3′

Lowercase characters of the forward primer represent the coding regionof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while bold characters represent a regionhomologous to insertion sites of pCaHj505 (WO2013029496). The 4 lettersahead of the coding sequence in the forward primer represent the Kozarksequence for initiation of translation.

Twenty picomoles of the forward and reverse primers above were used in aPCR composed of 1 μl of genomic DNA of Hamigera sp. strain NN046817, 10μl of 5× Phusion® HF Buffer (Finnzymes Oy, Espoo, Finland), 1.5 μl ofDMSO, 1.5 μl of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unitof PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland)in a final volume of 50 μl. The PCR was performed using a thermocyclerprogrammed for denaturing at 98° C. for 1 minute; 8 cycles of denaturingeach at 98° C. for 30 seconds, annealing at 66° C. for 30 seconds, witha 1° C. decrease per cycle and elongation at 72° C. for 2 minutes; 25cycles each at 98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C.for 2 minutes; and a final extension at 72° C. for 7 minutes. The heatblock then went to a 4° C. soak cycle.

The PCR product was isolated by 1.0% agarose gel electrophoresis using90 mM Tris-borate and 1 mM EDTA (TBE) buffer where a single product bandof approximately 1.6 kb was visualized under UV light. The PCR solutionwas then treated with the Cloning Enhancer (Clontech Laboratories, Inc.)by adding 2 μl of the Cloning Enhancer to 5 μl of the PCR solution. ThePCR solution was incubated at 37° C. for 20 minutes and then at 80° C.for 15 minutes in a thermocycler.

Plasmid pCaHj505 was digested with Barn HI and Xho I, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare)according to the manufacturer's instructions.

The Cloning Enhancer treated PCR solution and digested plasmid pCaHj505were ligated together using an In-Fusion® HD Cloning Kit (ClontechLaboratories, Inc.) according to the manufacturer's instructionsresulting in plasmid p505-GH7_Hami (FIG. 1), in which transcription ofthe Hamigera sp. GH7 cellobiohydrolase I polypeptide coding sequence wasunder the control of an Aspergillus oryzae alpha-amylase gene promoter.In brief, 1 μl of 30 ng/ul of pCaHj505, digested with Barn HI and Xho I,and 3 μl of the Cloning Enhancer treated PCR solution containing ˜60 ngof the Hamigera sp. GH7 cellobiohydrolase I PCR fragment were added to 1μl of 5× In-Fusion HD Enzyme Premix. The reaction was incubated at 37°C. for 15 minutes and then 50° C. for 15 minutes. Three μl of theligation reaction were used to transform E. coli TOP10 competent cells(TIANGEN Biotech Co. Ltd.). E. coli transformants containing anexpression construct were detected by colony PCR. Colony PCR is a methodfor quick screening of plasmid inserts directly from E. coli colonies.Briefly, a single colony was transferred to a premixed PCR solution in aPCR tube, including PCR buffer, MgCl₂, dNTPs, and primer pairs fromwhich the PCR fragment was generated. Several colonies were screened.After the PCR, the reactions were analyzed by 1.0% agarose gelelectrophoresis using TBE buffer. Plasmid DNA was prepared using aQIAPREP® Spin Miniprep Kit (QIAGEN GmbH) from the colony showing aninsert with the expected size. The Hamigera sp. GH7 cellobiohydrolase Icoding sequence inserted in p505-GH7_Hami was confirmed by DNAsequencing using 3730XL DNA Analyzers (Applied Biosystems Inc.).

Example 4 Expression of Hamigera sp. GH7 Cellobiohydrolase I Gene inAspergillus oryzae

Aspergillus oryzae strain MT3568 was used for heterologous expression ofthe coding sequence of Hamigera sp. GH7 cellobiohydrolase I. A. oryzaeMT3568 is an amdS (acetamidase) disrupted gene derivative of A. oryzaeJaL355 (WO 02/40694) in which pyrG auxotrophy was restored by disruptingthe A. oryzae acetamidase (amdS) gene with the pyrG gene.

Protoplasts were prepared according to the method described as“Transformation of Aspergillus Expression Host” in Example 2 ofUS20140179588 A1. Three μg of p505-GH7_ Hami were used to transformAspergillus oryzae MT3568, which yielded about 50 transformants. Fourtransformants were isolated to individual Minimal medium plates and werethen inoculated separately into 3 ml of YPM medium in 24-well plate andincubated at 30° C., 150 rpm. After 3 days incubation, 20 μl ofsupernatant from each culture were analyzed on NuPAGE Novex 4-12%Bis-Tris Gel w/MES (Invitrogen Corporation) according to themanufacturer's instructions. The resulting gel was stained with InstantBlue (Expedeon Ltd.). SDS-PAGE profiles of the cultures showed that themajority of the transformants had a major band of approximately 55 kDa.The expression strain was designated A. oryzae O521DH.

Example 5 Fermentation of Expression Strain Aspergillus oryzae O521DH

A slant of the expression strain A. oryzae O521DH was washed with 10 mlof YPM medium and inoculated into four 2-liter flasks each containing400 ml of YPM medium to generate broth for purification andcharacterization of the Hamigera sp. GH7 cellobiohydrolase I. Theculture was harvested on day 3 and filtered using a 0.45 μm DURAPOREMembrane (Millipore).

Example 6 Purification of Recombinant Hamigera sp. CBHI from Aspergillusoryzae O521DH

A 1600 ml volume of filtered supernatant of Aspergillus oryzae O521DH(Example 5) was precipitated with ammonium sulfate (80% saturation),re-dissolved in 50 ml of 20 mM sodium acetate pH 5.5, dialyzed againstthe same buffer, and filtered through a 0.45 μm filter. The final volumewas 60 ml. The solution was applied to a 40 ml Q SEPHAROSE® Fast Flowcolumn (GE Healthcare) equilibrated with 20 mM sodium acetate pH 5.5.Proteins were eluted with a linear 0-0.25 M NaCl gradient. Fractionsunbound to the column were collected and further purified using a 40 mlPhenyl SEPHAROSE® 6 Fast Flow column (GE Healthcare) with a linear 1.2-0M ammonium sulfate gradient. Fractions were analyzed by SDS-PAGE using aNUPAGE® NOVEX® 4-12% Bis-Tris Gel (Invitrogen Corporation) with 50 mMMES. The resulting gel was stained with INSTANTBLUE™ (Expedeon Ltd.).Fractions containing a band at approximately 55 kDa were pooled. Thenthe pooled solution was concentrated by ultrafiltration.

Example 7 Preparation of Enzyme Compositions

A base enzyme composition designated “cellulolytic enzyme compositionwithout cellobiohydrolase I” was prepared composed of 30% Aspergillusfumigatus GH6 cellobiohydrolase II, 10% Trichoderma reesei GH5endoglucanase II, 10% Trichoderma reesei GH7 endoglucanase I, 3%Penicillium emersonii GH61A polypeptide, 4% Aspergillus fumigatus GH10xylanase, 36% Aspergillus fumigatus beta-glucosidase, and 7% Aspergillusfumigatus beta-xylosidase.

The Aspergillus fumigatus GH6A cellobiohydrolase II (GEN ESEQP:AZI04854)was prepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The Aspergillus fumigatus GH6A cellobiohydrolase II waspurified according to W/O 2012/122518.

The Trichoderma reesei GH5 endoglucanase II (GENESEQP:AZI04858) wasprepared recombinantly according to WO 2011/057140 using Aspergillusoryzae as a host. The filtered broth of the T. reesei endoglucanase IIwas desalted and buffer-exchanged into 20 mM Tris pH 8.0 using atangential flow concentrator (Pall Filtron) equipped with a 10 kDapolyethersulfone membrane (Pall Filtron).

The Trichoderma reesei GH7 endoglucanase I (GENESEQP:ARV30516) wasprepared recombinantly according to WO 2005/067531 using Aspergillusoryzae as a host. To purify the Trichoderma reesei GH7 endoglucanase I,the filtered broth was diluted to 20 mM Tris pH 8.0 with 0.75 M ammoniumsulfate. The protein was applied to a Phenyl Sepharose™ High Performancecolumn (GE Healthcare) equilibrated with 20 mM Tris pH 8.0 with 1 Mammonium sulfate and bound proteins were eluted with a linear gradientfrom 1 to 0 M ammonium sulfate. Pooled fractions were concentrated anddesalted using a VIVASPIN™ 20 centrifugal concentrator with a molecularweight cut-off of 10 kDa (Sartorius Stedim Biotech S.A.) into 50 mMsodium acetate pH 5.0 containing 150 mM sodium chloride.

The Penicillium sp. GH61A polypeptide (GENESEQP: AZI04878) was preparedrecombinantly according to W/O 20111/057140 using Trichoderma reesei asa host. To purify the Penicillium sp. GH61A polypeptide, a fermentationculture medium was desalted using a tangential flow concentrator (PallFiltron) equipped with a 10 kDa polyethersulfone membrane (Pall Filtron)into 20 mM Tris-HCl pH 8.5. The buffer-exchanged sample was loaded ontoa Q SEPHAROSE® Fast Flow column (GE Healthcare) pre-equilibrated with 20mM Tris-HCl, pH 8.0, eluted with 20 mM Tris-HCl pH 8.0 and 1 M NaCl.Selected fractions were pooled and ammonium sulfate was added to 0.85 M,and then loaded onto a Phenyl SEPHAROSE® Fast Flow column (GEHealthcare) preequilibrated with 20 mM Tris-HCl, pH 7.5 and 0.85 Mammonium sulfate, eluted with 20 mM Tris-HCl, pH 7.5. The fractions werepooled and desalted using a tangential flow concentrator (Pall Filtron)equipped with a 10 kDa polyethersulfone membrane into 50 mM sodiumacetate pH 5.0.

The Aspergillus fumigatus GH10 xylanase (GENESEQP:AZI04884) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2(WO 2000/39322) as a host. The filtered broth of the A. fumigatusxylanase was desalted and buffer-exchanged into 50 mM sodium acetate pH5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare).

The Aspergillus fumigatus Cel3A beta-glucosidase 4M variant(GENESEQP:AZU67153) was recombinantly prepared according to WO2012/044915. The filtered broth of Aspergillus fumigatus Cel3Abeta-glucosidase 4M was concentrated and buffer exchanged using atangential flow concentrator (Pall Filtron) equipped with a 10 kDapolyethersulfone membrane (Pall Filtron) with 50 mM sodium acetate pH5.0 containing 100 mM sodium chloride. Protein concentration wasdetermined using 4-nitrophenyl-beta-D-glucopyranoside (Sigma ChemicalCo.) as a substrate and Aspergillus fumigatus Cel3A beta-glucosidase 4M280 as a protein standard purified according to WO 2012/044915 with theprotein concentration determined using the theoretic extinctioncoefficient and the absorbance of the protein at 280 nm. The4-nitrophenyl-beta-D-glucopyranoside (pNPG) was performed as follows:pNPG was dissolved in DMSO to make 100 mM stock solution. The 100 mMpNPG stock solution was diluted 100× in 50 mM sodium acetate buffer pH 5with 0.01% TWEEN® 20 to 1 mM pNPG containing 50 mM sodium acetate bufferpH 5 with 0.01% TWEEN® 20. The protein was diluted to severalconcentrations in 50 mM sodium acetate buffer pH 5 with 0.01% TWEEN® 20.Then, 20 μl of the diluted protein were added to 100 μl of 1 mM pNPGcontaining 50 mM sodium acetate buffer pH 5 with 0.01% TWEEN® 20. Thereactions were incubated at 40° C. for 20 minutes, and then stopped with50 μl 1M sodium carbonate buffer pH 10. The absorbance was measured forpNP production at 405 nm.

The Aspergillus fumigatus GH3 beta-xylosidase (GENESEQP:AZI05042) wasprepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the A. fumigatus GH3 beta-xylosidasewas desalted and buffer-exchanged into 50 mM sodium acetate pH 5.0 usinga HIPREP® 26/10 Desalting Column (GE Healthcare).

The protein concentration for each of the monocomponents described abovewas determined using A₂₈₀.

Example 8 Preparation of Hamigera sp. Cellobiohydrolase I andAspergillus fumigatus Cellobiohydrolase I

The Hamigera sp. cellobiohydrolase I was prepared according to themethod described in example 6.

The Aspergillus fumigatus Cel7A cellobiohydrolase I (GENESEQP: AZI04842)was prepared recombinantly in Aspergillus oryzae as described in WO2011/057140. To purify the Aspergillus fumigatus Cel7A cellobiohydrolaseI, a fermentation culture medium was desalted using a tangential flowconcentrator (Pall Filtron) equipped with a 10 kDa polyethersulfonemembrane (Pall Filtron) into 20 mM Tris-HCl pH 8.0. The desaltedmaterial was loaded onto a Q SEPHAROSE® High Performance column (GEHealthcare) equilibrated in 20 mM Tris pH 8.0, and bound proteins wereeluted with a linear gradient from 0-600 mM sodium chloride. Poolfractions were combined.

The protein concentration for each of the components described above wasdetermined using A₂₈₀.

Example 9 Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 0.048 g sulfuric acid per g drybiomass at 190° C. and 25% w/w dry solids for approximately 1 minute.The water-insoluble solids in the pretreated corn stover (PCS) contained5653.2% cellulose, 3.6% hemicellulose and 29.8% lignin. Cellulose andhemicellulose were determined by a two-stage sulfuric acid hydrolysiswith subsequent analysis of sugars by high performance liquidchromatography using NREL Standard Analytical Procedure #002. Lignin wasdetermined gravimetrically after hydrolyzing the cellulose andhemicellulose fractions with sulfuric acid using NREL StandardAnalytical Procedure #003.

Prior to enzymatic hydrolysis, the PCS was ground using a COSMOS® MultiUtility Grinder (EssEmm Corporation), sieved through 420 μm sieve, andautoclaved at 121° C. for 30 minutes. The dry content of the ground andsieved PCS was 3.84%.

Hydrolysis of the ground/sieved PCS was performed using the cellulolyticenzyme composition without cellobiohydrolase I (Example 7) at 1.5 mgenzyme/g total solids, supplemented with either Hamigera sp.cellobiohydrolase I or Aspergillus fumigatus cellobiohydrolase I at 0.4,0.5, 0.6, or 0.7 mg enzyme/g total solids, at 50° C., pH 5.0. Totalinsoluble solids loading of the PCS was 31 g/L (in 50 mM sodium acetatepH 5.0 buffer containing 1 mM manganese sulfate). The total reactionvolume was 0.25 ml in 96-well plates (Fisher Scientific). Assays wererun in triplicate. After 72 hours of incubation at 50° C., supernatantswere taken and filtered through a 0.45 μm 96-well filter plate(Millipore), diluted 2-fold in 5 mM H₂SO₄, and analyzed using a AgilentHPLC (Agilent Technologies) equipped with an Aminex HPX-87H column(Bio-Rad Laboratories, Inc.) and refractive index detection. Hydrolysisdata are presented as % of total cellulose converted to glucose. Thedegree of cellulose conversion to reducing sugar was calculated usingthe following equation:

$\begin{matrix}{{Conversion}_{(\%)} = {{RS}_{({m\;{g/m}\; l})}*100*{162/\left( {{Cellulose}_{({m\;{g/m}\; l})}*180} \right)}}} \\{= {{RS}_{({m\;{g/m}\; l})}*{100/\left( {{Cellulose}_{({m\;{g/m}\; l})}*1.111} \right)}}}\end{matrix}$

RS is the concentration of reducing sugar in solution measured inglucose equivalents (mg/ml), and the factor 1.111 reflects the weightgain in converting cellulose to glucose. Table 1 showed that in NREL PCShydrolysis, Hamigera sp. cellobiohydrolase I outperformed theAspergillus fumigatus cellobiohydrolase I at 50° C., pH 5.0.

TABLE 1 Conversion by two CBHI enzymes in PCS hydrolysis, 50° C., pH5.0. Cellobiohydrolase I Hamigera sp. Aspergillus fumigatus loading,mg/g TS cellobiohydrolase I cellobiohydrolase I 0  0%  0% 0.4 41% 24%0.5 46% 28% 0.6 49% 31% 0.7 52% 34%

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The present invention is further described by the followed numberedparagraphs:

-   1. A polypeptide having cellobiohydrolase activity, selected from    the group consisting of:

(a) a polypeptide having at least 82% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the full-length complement of (i);

(c) a polypeptide encoded by a polynucleotide having at least 82%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellobiohydrolase activity.

-   2. The polypeptide of paragraph 1, having at least 82%, at least    83%, at least 84%, at least 85%, at least 86%, at least 87%, at    least 88%, at least 89%, at least 90%, at least 91%, at least 92%,    at least 93%, at least 94%, at least 95%, at least 96%, at least    97%, at least 98%, at least 99% or 100% sequence identity to the    mature polypeptide of SEQ ID NO: 2.-   3. The polypeptide of paragraph 1 or 2, which is encoded by a    polynucleotide that hybridizes under high stringency conditions or    very high stringency conditions with (i) the mature polypeptide    coding sequence of SEQ ID NO: 1, (ii) the full-length complement of    (i).-   4. The polypeptide of any of paragraphs 1-3, which is encoded by a    polynucleotide having at least 82%, at least 83%, at least 84%, at    least 85%, at least 86%, at least 87%, at least 88%, at least 89%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide coding    sequence of SEQ ID NO: 1.-   5. The polypeptide of any of paragraphs 1-4, comprising or    consisting of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO:    2.-   6. The polypeptide of paragraph 5, wherein the mature polypeptide is    amino acids 26 to 525 of SEQ ID NO: 2.-   7. The polypeptide of any of paragraphs 1-4, which is a variant of    the mature polypeptide of SEQ ID NO: 2 comprising a substitution,    deletion, and/or insertion at one or more positions.-   8. The polypeptide of paragraph 1, which is a fragment of SEQ ID NO:    2, wherein the fragment has cellobiohydrolase activity.-   9. A polypeptide comprising a catalytic domain selected from the    group consisting of:

(a) a catalytic domain having at least 82% sequence identity to aminoacids 26 to 466 of SEQ ID NO: 2;

(b) a catalytic domain encoded by a polynucleotide that hybridizes underhigh stringency conditions with (i) nucleotides 76 to 1398 of SEQ ID NO:1, (ii) the full-length complement of (i);

(c) a catalytic domain encoded by a polynucleotide having at least 82%sequence identity to the catalytic domain of SEQ ID NO: 1;

(d) a variant of amino acids 26 to 466 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hascellobiohydrolase activity.

-   10. The polypeptide of paragraph 9, further comprising a    carbohydrate binding module.-   11. A polypeptide comprising a carbohydrate binding module operably    linked to a catalytic domain, wherein the binding module is selected    from the group consisting of:

(a) a carbohydrate binding module having at least 81% sequence identityto amino acids 489 to 525 of SEQ ID NO: 2;

(b) a carbohydrate binding module encoded by a polynucleotide thathybridizes under high stringency conditions with (i) nucleotides 1465 to1575 of SEQ ID NO: 1, (ii) the full-length complement of (i);

(c) a carbohydrate binding module encoded by a polynucleotide having atleast 82% sequence identity to nucleotides 1465 to 1575 of SEQ ID NO: 1;

(d) a variant of amino acids 489 to 525 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and

(e) a fragment of (a), (b), (c), (d) or (e) that has carbohydratebinding activity.

-   12. The polypeptide of paragraph 11, wherein the catalytic domain is    obtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase,    or transferase, e.g., an aminopeptidase, amylase, carbohydrase,    carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,    cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,    endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,    glucoamylase, alpha-glucosidase, beta-glucosidase, invertase,    laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic    enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,    ribonuclease, transglutaminase, xylanase, or beta-xylosidase.-   13. A composition comprising the polypeptide of any of paragraphs    1-12.-   14. A whole broth formulation or cell culture composition comprising    the polypeptide of any of paragraphs 1-12.-   15. A polynucleotide encoding the polypeptide of any of paragraphs    1-12.-   16. A nucleic acid construct or expression vector comprising the    polynucleotide of paragraph 15 operably linked to one or more    control sequences that direct the production of the polypeptide in    an expression host.-   17. A recombinant host cell comprising the polynucleotide of    paragraph 15 operably linked to one or more control sequences that    direct the production of the polypeptide.-   18. A method of producing the polypeptide of any of paragraphs 1-12,    comprising cultivating a cell, which in its wild-type form produces    the polypeptide, under conditions conducive for production of the    polypeptide.-   19. The method of paragraph 17, further comprising recovering the    polypeptide.-   20. A method of producing a polypeptide having cellobiohydrolase    activity, comprising cultivating the host cell of paragraph 16 under    conditions conducive for production of the polypeptide.-   21. The method of paragraph 19, further comprising recovering the    polypeptide.-   22. A transgenic plant, plant part or plant cell transformed with a    polynucleotide encoding the polypeptide of any of paragraphs 1-12.-   23. A method of producing a polypeptide having cellobiohydrolase    activity, comprising cultivating the transgenic plant or plant cell    of paragraph 22 under conditions conducive for production of the    polypeptide.-   24. The method of paragraph 23, further comprising recovering the    polypeptide.-   25. A process for degrading a cellulosic or hemicellulosic material,    comprising: treating the cellulosic or hemicellulosic material with    an enzyme composition comprising the polypeptide having    cellobiohydrolase activity of any of paragraphs 1-11.-   26. The process of paragraph 25, wherein the cellulosic or    hemicellulosic material is pretreated.-   27. The process of paragraph 25 or 26, wherein the enzyme    composition further comprises one or more enzymes selected from the    group consisting of a cellulase, an AA9 polypeptide, a    hemicellulase, a cellulose inducible protein (CIP) an esterase, an    expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a    protease, and a swollenin.-   28. The process of paragraph 27, wherein the cellulase is one or    more enzymes selected from the group consisting of an endoglucanase,    a cellobiohydrolase, and a beta-glucosidase.-   29. The process of paragraph 27, wherein the hemicellulase is one or    more enzymes selected from the group consisting of a xylanase, an    acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a    xylosidase, and a glucuronidase.-   30. The process of any of paragraphs 25-29, further comprising    recovering the degraded cellulosic or hemicellulosic material.-   31. The process of paragraph 30, wherein the degraded cellulosic or    hemicellulosic material is a sugar.-   32. The process of paragraph 31, wherein the sugar is selected from    the group consisting of glucose, xylose, mannose, galactose, and    arabinose.-   33. A process for producing a fermentation product, comprising:

(a) saccharifying a cellulosic or hemicellulosic material with an enzymecomposition comprising the polypeptide having cellobiohydrolase activityof any of paragraphs 1-9;

(b) fermenting the saccharified cellulosic or hemicellulosic materialwith one or more fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

-   34. The process of paragraph 33, wherein the cellulosic or    hemicellulosic material is pretreated.-   35. The process of paragraph 32 or 34, wherein the enzyme    composition further comprises one or more enzymes selected from the    group consisting of a cellulase, an AA9 polypeptide, a    hemicellulase, a CIP, an esterase, an expansin, a ligninolytic    enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.-   36. The process of paragraph 35, wherein the cellulase is one or    more enzymes selected from the group consisting of an endoglucanase,    a cellobiohydrolase, and a beta-glucosidase.-   37. The process of paragraph 35 or 36, wherein the hemicellulase is    one or more enzymes selected from the group consisting of a    xylanase, an acetylxylan esterase, a feruloyl esterase, an    arabinofuranosidase, a xylosidase, and a glucuronidase.-   38. The process of any of paragraphs 33-37, wherein steps (a)    and (b) are performed simultaneously in a simultaneous    saccharification and fermentation.-   39. The process of any of paragraphs 33-38, wherein the fermentation    product is an alcohol, an alkane, a cycloalkane, an alkene, an amino    acid, a gas, isoprene, a ketone, an organic acid, or polyketide.-   40. A process of fermenting a cellulosic or hemicellulosic material,    comprising: fermenting the cellulosic or hemicellulosic material    with one or more fermenting microorganisms, wherein the cellulosic    or hemicellulosic material is saccharified with an enzyme    composition and the polypeptide having cellobiohydrolase activity of    any of paragraphs 1-9.-   41. The process of paragraph 40, wherein the cellulosic or    hemicellulosic material is pretreated before saccharification.-   42. The process of paragraph 40 or 41, wherein the enzyme    composition further comprises one or more enzymes selected from the    group consisting of a cellulase, an AA9 polypeptide, a    hemicellulase, a CIP, an esterase, an expansin, a ligninolytic    enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.-   43. The process of paragraph 42, wherein the cellulase is one or    more enzymes selected from the group consisting of an endoglucanase,    a cellobiohydrolase, and a beta-glucosidase.-   44. The process of paragraph 42, wherein the hemicellulase is one or    more enzymes selected from the group consisting of a xylanase, an    acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a    xylosidase, and a glucuronidase.-   45. The process of any of paragraphs 40-44, wherein the fermenting    of the cellulosic or hemicellulosic material produces a fermentation    product.-   46. The process of paragraph 45, further comprising recovering the    fermentation product from the fermentation.-   47. The process of paragraph 45 or 46, wherein the fermentation    product is an alcohol, an alkane, a cycloalkane, an alkene, an amino    acid, a gas, isoprene, a ketone, an organic acid, or polyketide.

What is claimed is:
 1. A nucleic acid construct comprising apolynucleotide encoding a polypeptide having cellobiohydrolase activity,wherein the polynucleotide is operably linked to one or moreheterologous control sequences that direct production of the polypeptidein an expression host, and wherein the polypeptide is selected from: (a)a polypeptide having at least 90% sequence identity to amino acids 26 to525 of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide thathybridizes under high stringency conditions with the full-lengthcomplement of nucleotides 76 to 1575 of SEQ ID NO: 1; and (c) apolypeptide encoded by a polynucleotide having at least 90% sequenceidentity to nucleotides 76 to 1575 of SEQ ID NO:
 1. 2. A nucleic acidconstruct comprising a polynucleotide encoding a polypeptide havingcellobiohydrolase activity, wherein the polynucleotide is operablylinked to one or more heterologous control sequences that directproduction of the polypeptide in an expression host, and wherein thepolypeptide comprises a catalytic domain selected from: (a) a catalyticdomain having at least 90% sequence identity to amino acids 26 to 466 ofSEQ ID NO: 2; (b) a catalytic domain encoded by a polynucleotide thathybridizes under high stringency conditions with the full-lengthcomplement of nucleotides 76 to 1398 of SEQ ID NO: 1; and (c) acatalytic domain encoded by a polynucleotide having at least 90%sequence identity to nucleotides 76 to 1398 of SEQ ID NO:
 1. 3. Thenucleic acid construct of claim 2, wherein the polypeptide furthercomprises a carbohydrate binding module.
 4. A nucleic acid constructcomprising a polynucleotide encoding a polypeptide, wherein thepolynucleotide is operably linked to one or more heterologous controlsequences that direct production of the polypeptide in an expressionhost, wherein the polypeptide comprises a carbohydrate binding moduleoperably linked to a catalytic domain, and wherein the carbohydratebinding module is selected from: (a) a carbohydrate binding modulehaving at least 90% sequence identity to amino acids 489 to 525 of SEQID NO: 2; (b) a carbohydrate binding module encoded by a polynucleotidethat hybridizes under high stringency conditions with the full-lengthcomplement of nucleotides 1465 to 1575 of SEQ ID NO: 1; and (c) acarbohydrate binding module encoded by a polynucleotide having at least90% sequence identity to nucleotides 1465 to 1575 of SEQ ID NO:
 1. 5.The nucleic acid construct of claim 4, wherein the catalytic domain isobtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase.
 6. A recombinant host cell comprising the nucleic acidconstruct of claim
 1. 7. A method of producing a polypeptide havingcellobiohydrolase activity, comprising cultivating the host cell ofclaim 6 under conditions conducive for production of the polypeptide. 8.The method of claim 7, further comprising recovering the polypeptide. 9.A method of degrading or converting a cellulosic material, said methodcomprising: (i) treating the cellulosic material with a compositioncomprising a polypeptide having cellobiohydrolase activity; and (ii)recovering the degraded or converted cellulosic material; wherein thepolypeptide having cellobiohydrolase activity is selected from: (a) apolypeptide having at least 90% sequence identity to amino acids 26 to525 of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide thathybridizes under high stringency conditions with the full-lengthcomplement of nucleotides 76 to 1575 of SEQ ID NO: 1; and (c) apolypeptide encoded by a polynucleotide having at least 90% sequenceidentity to nucleotides 76 to 1575 of SEQ ID NO:
 1. 10. The method ofclaim 9, wherein the polypeptide has at least 95% sequence identity withamino acids 26 to 525 of SEQ ID NO:
 2. 11. The method of claim 9,wherein the polypeptide has at least 97% sequence identity with aminoacids 26 to 525 of SEQ ID NO:
 2. 12. The method of claim 9, wherein thepolypeptide having cellobiohydrolase activity comprises or consists ofamino acids 26 to 525 of SEQ ID NO:
 2. 13. A method of degrading orconverting a cellulosic material, said method comprising: (i) treatingthe cellulosic material with a composition comprising a polypeptidehaving cellobiohydrolase activity; and (ii) recovering the degraded orconverted cellulosic material; wherein the polypeptide havingcellobiohydrolase activity comprises a catalytic domain selected from:(a) a catalytic domain having at least 90% sequence identity to aminoacids 26 to 466 of SEQ ID NO: 2; (b) a catalytic domain encoded by apolynucleotide that hybridizes under high stringency conditions with thefull-length complement of nucleotides 76 to 1398 of SEQ ID NO: 1; and(c) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to the catalytic domain of SEQ ID NO:
 1. 14. Themethod of claim 13, wherein the catalytic domain has at least 95%sequence identity with amino acids 26 to 466 of SEQ ID NO:
 2. 15. Themethod of claim 13, wherein the catalytic domain has at least 97%sequence identity with amino acids 26 to 466 of SEQ ID NO:
 2. 16. Themethod of claim 13, wherein the catalytic domain comprises or consistsof amino acid 26 to 466 of SEQ ID NO:
 2. 17. The nucleic acid constructof claim 1, wherein the polypeptide has at least 95% sequence identitywith amino acids 26 to 525 of SEQ ID NO:
 2. 18. The nucleic acidconstruct of claim 1, wherein the polypeptide has at least 97% sequenceidentity with amino acids 26 to 525 of SEQ ID NO:
 2. 19. The nucleicacid construct of claim 1, wherein the polypeptide havingcellobiohydrolase activity comprises or consists of amino acid 26 to 525of SEQ ID NO:
 2. 20. The nucleic acid construct of claim 2, wherein thecatalytic domain has at least 95% sequence identity with amino acids 26to 466 of SEQ ID NO:
 2. 21. The nucleic acid construct of claim 2,wherein the catalytic domain has at least 97% sequence identity withamino acids 26 to 466 of SEQ ID NO:
 2. 22. The nucleic acid construct ofclaim 2, wherein the catalytic domain comprises or consists of aminoacids 26 to 466 of SEQ ID NO:
 2. 23. The nucleic acid construct of claim4, wherein the carbohydrate binding module has at least 95% sequenceidentity with amino acids 489 to 525 of SEQ ID NO:
 2. 24. The nucleicacid construct of claim 4, wherein the carbohydrate binding module hasat least 97% sequence identity with amino acids 489 to 525 of SEQ ID NO:2.
 25. The nucleic acid construct of claim 4, wherein the carbohydratebinding module comprises or consists of amino acids 489 to 525 of SEQ IDNO:
 2. 26. The method of claim 7, wherein the polypeptide has at least95% sequence identity with amino acids 26 to 525 of SEQ ID NO:
 2. 27.The method of claim 7, wherein the polypeptide has at least 97% sequenceidentity with amino acids 26 to 525 of SEQ ID NO:
 2. 28. The method ofclaim 7, wherein the polypeptide having cellobiohydrolase activitycomprises or consists of amino acids 26 to 525 of SEQ ID NO:
 2. 29. Arecombinant host cell transformed with a nucleic acid construct orexpression vector comprising a polynucleotide encoding a polypeptidehaving cellobiohydrolase activity, wherein the polypeptide havingcellobiohydrolase activity is heterologous to the recombinant host cell,and wherein the polypeptide having cellobiohydrolase activity comprisesan amino acid sequence which has at least 90% sequence identity withamino acids 26 to 525 of SEQ ID NO:
 2. 30. The recombinant host cell ofclaim 29, wherein the polypeptide having cellobiohydrolase activitycomprises an amino acid sequence which has at least 95% sequenceidentity with amino acids 26 to 525 of SEQ ID NO:
 2. 31. The recombinanthost cell of claim 29, wherein the polypeptide having cellobiohydrolaseactivity comprises an amino acid sequence which has at least 97%sequence identity with amino acids 26 to 525 of SEQ ID NO:
 2. 32. Therecombinant host cell of claim 29, wherein the polypeptide havingcellobiohydrolase activity comprises or consists of the amino acidsequence of amino acids 26 to 525 of SEQ ID NO: 2.